Differences in drug metabolism pathways across species necessitate the use of in vitro human liver models for screening the metabolism and toxicity of drugs/industrial chemicals, and for discovering novel therapeutics against liver diseases. While primary human hepatocytes (PHHs) are the ?gold standard? for fabricating human liver models, a severe shortage of donor livers limits their utility for high-throughput screening and prevents a comprehensive assessment of genetic determinants of diseases. In contrast, induced pluripotent stem cell- derived human hepatocyte-like cells (iPSC-HHs) created from genetically diverse donor panels can address the limitations of PHHs; however, the protocols to generate iPSC-HHs cannot fully mature these cells towards an adult PHH phenotype and thus such protocols require substantial refinement. An enhanced understanding of how liver-inspired microenvironmental cues synergistically affect iPSC-HH maturation and maintenance of phenotype is urgently needed to utilize iPSC-HHs more effectively for the above applications. The adult liver contains an extracellular matrix (ECM) that a) presents biochemical and biomechanical signals, and b) can further modulate interactions of liver cells with growth factors and cytokines. Previous investigations have demonstrated that the composition of ECM, substrate stiffness, and growth factors can independently influence the in vitro functions of primary hepatocytes and embryonic stem cell-derived hepatocyte-like cells. However, the crosstalk between these cues as occurs in vivo is unclear, and the combinatorial effects of ECM and growth factor signals on the functional maturation of iPSC-HHs have yet to be identified. In addition, the liver has several non-parenchymal cell (NPC) types that can influence hepatocellular functions, including liver sinusoidal endothelial cells (LSECs) that provide critical regulatory signals to hepatocytes during liver development, physiology, and regeneration; our recent work also supports the notion that LSECs regulate iPSC-HH functions to a greater degree than other liver NPC types. Thus, in this proposal we will examine the central hypothesis that ECM composition/stiffness and LSEC intercellular interactions collectively act to significantly modulate iPSC-HH functional maturity. Part of the challenge in testing such a hypothesis is that evaluating a large number of combinations of microenvironmental cues in a bulk culture format is too costly as well as labor and time intensive. Therefore, we will adapt a cellular microarray platform that enables defined ECM microenvironments, combinatorial culture with soluble factors, and quantitative assessment of cell phenotype using high-content imaging readouts. In Aim 1, we will systematically investigate the cooperative effects of ECM composition, substrate stiffness, and growth factor signaling on iPSC-HH phenotype. In Aim 2, we will examine reciprocal interactions between iPSC-HHs and LSECs and establish a platform for drug toxicity assessment. Collectively, these studies are essential for further developing in vitro liver models containing iPSC-HHs for clinically-predictive drug/chemical screening, and ultimately, regenerative medicine.